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Abstract

Background

Short-chain fructo-oligosaccharides (scFOS) are increasingly used in human diet for
their prebiotic properties. We aimed at investigating the effects of scFOS ingestion
on the colonic microflora and oro-fecal transit time in elderly healthy humans.

Methods

Stools composition, oro-fecal transit time, and clinical tolerance were evaluated
in 12 healthy volunteers, aged 69 ± 2 yrs, in three consecutive periods: basal period
(2 weeks), scFOS (Actilight®) ingestion period (8 g/d for 4 weeks) and follow-up period (4 weeks). Two-way ANOVA,
with time and treatment as factors, was used to compare the main outcome measures
between the three periods.

Results

Fecal bifidobacteria counts were significantly increased during the scFOS period (9.17
± 0.17 log cfu/g vs 8.52 ± 0.26 log cfu/g during the basal period) and returned to
their initial values at the end of follow-up (8.37 ± 0.21 log cfu/g; P < 0.05). Fecal
cholesterol concentration increased during the scFOS period (8.18 ± 2.37 mg/g dry
matter vs 2.81 ± 0.94 mg/g dry matter during the basal period) and returned to the
baseline value at the end of follow-up (2.87 ± 0.44 mg/g dry matter; P < 0.05). Fecal
pH tended to decrease during scFOS ingestion and follow-up periods compared to the
basal period (P = 0.06). Fecal bile acids, stool weight, water percentage, and oro-fecal
transit time did not change throughout the study. Excess flatus and bloating were
significantly more frequent during scFOS ingestion when compared to the basal period
(P < 0.05), but the intensity of these symptoms was very mild.

Conclusion

Four-week 8 g/d scFOS ingestion is well tolerated and leads to a significant increase
in fecal bifidobacteria in healthy elderly subjects. Whether the change in cholesterol
metabolism found in our study could exert a beneficial action warrants further studies.

Background

Short-chain fructo-oligosaccharides (scFOS) are a mixture of oligosaccharides consisting
of glucose linked to fructose units [1]. They are poorly absorbed in the human small intestine [2], but are fermented in the colon by the resident microflora [3]. It is now well established that scFOS meet criteria to be considered as prebiotic,
defined as a non digestible food ingredient that beneficially affects the host by
selectively stimulating growth and/or activity of one or a limited number of colonic
bacteria, and thus improves host health [4]. We have shown in humans that dietary addition of 10 g/d scFOS led to increasing
fecal counts of bifidobacteria [5]; moreover, the scFOS administration dose-dependently increases fecal bifidobacteria
in healthy volunteers, with an optimal and well-tolerated dose ranging from 2.5 to
10 g/d [6,7].

Bifidobacteria are considered beneficial to health [8], even if sound evidence of such effect is not available yet [4]. In mice, in vivo administered bifidobacteria along with fructo-oligosaccharides reduced 1,2-dimethylhydrazine
induced carcinogenesis [9]. In rats, Bifidobacterium longum, administered alone or in association with non-digestible oligosaccharides, exerts
strong antitumour activity [10,11] This effect could be due to colon acidification that inhibits bacterial degradation
of primary to carcinogenic secondary bile acids [12] and/or to increasing bifidobacteria population. Indeed, bifidobacteria per se could have an anti-tumorigenic activity. Bifidobacteria reduce nitrosamine mutagenicity,
and Bifidobacterium bifidum administered along with Lactobacillus acidophilus to healthy humans decreases nitroreductase activity in stools [13]. Lastly, oligosaccharide ingestion could result in increasing colonic contents and
decreasing transit time [14], both factors may affect colonic carcinogen concentration and mucosal contact time
[15]. Thus, taking into account the intrinsic anti-tumoral properties of bifidobacteria
and the effects on colonic pH, fecal mass and transit time, a potential benefit of
scFOS ingestion could be colon cancer prevention, in particular in the elderly, who
are particularly at risk of developing colon cancer [16].

Although colonic microbiota is relatively stable throughout adult life, age-related
changes in the gastrointestinal tract inevitably affect its composition [17]. Bifidobacteria are numerically important colonic species that can be found in adults
[18], and the decline in bifidobacteria numbers is one of the most marked changes in the
elderly gut [19]. These changes, along with general reduction in species diversity in most bacterial
groups, as well as changes in diet and digestive physiology, such as intestinal transit
time, may result in increased putrefaction in the colon, and greater susceptibility
to disease. Dietary supplements containing prebiotics have been suggested to counteract
these changes in the elderly [20-22].

In that context, the aim of our study was to assess in healthy elderly the effects
of four-week scFOS ingestion on colonic microflora and oro-fecal transit time (OFTT).

Methods

Subjects

Twelve elderly healthy volunteers, six men and six women, aged 69 ± 2 years, participated
in the study. None of them had any gastrointestinal disease history. No antibiotics
or laxatives had been taken during the 3 months before the study. No other medication
was allowed during the investigation period. The subjects signed a written informed
consent to the protocol, which was approved by Lariboisière – Saint-Louis Hospital
Ethics Committee.

Study Design

The study was conducted in Saint-Lazare Hospital, Paris, France. It was divided into
three periods: basal (weeks 1–2), scFOS (weeks 3–6), and follow-up (weeks 7–10) periods.
Throughout the study, volunteers took their usual diet. Neither fermented dairy products
containing viable bifidobacteria and FOS (onions, asparagus, rye, and Jerusalem artichoke)
were allowed, nor food known to induce abdominal symptoms (beans, cabbage, raisin,
banana, and wheat bran). During scFOS period, subjects received 8 g/d scFOS in two
oral doses at the end of breakfast and diner. This dose was defined as a good compromise
between efficacy and tolerance. We used scFOS from Actilight® (Beghin Meiji, Marckolsheim, France), which consist of 44% 1-ketose (GF2), 46% nystose (GF3), and 10% 1F-β-fructofuranosyl nystose (GF4).

To measure the mean oro-fecal transit time (OFTT), the subjects ingested, with their
breakfast, 20 radio-opaque pellets of different shapes for three consecutive days
[23]. The first stools passed after the fourth day were collected, and their marker content
analysed. Stools had been collected for three consecutive days before the end of weeks
2 (basal period), 6 (ingestion period), and 10 (follow-up period), that is to say
at the end of each feeding period.

Tolerance to administered scFOS was evaluated using a daily chart in which the symptoms
(excessive flatus, borborygmi, bloating, and abdominal pain) were rated from zero
(no symptom) to three (severe symptom). Stool frequency and consistency were also
graded by the volunteers. Diarrhoea was defined as one or more watery stools, or more
than three stools per day.

Stool collection

Stools were collected three times, for 48 h at the end of each period (weeks 2, 6,
and 10). Samples were collected in plastic containers rendered anaerobic (Anaerocult
A; Merck, Darmstadt, Germany), immediately transferred to the laboratory, and then
analysed for bacterial counts and pH within 2 hours. Stools were then frozen at -20°C
for further analysis.

Bacterial counts and pH

Fresh faecal samples (1 g) were introduced in the first pre-weighed tube of the dilution
series and thoroughly mixed, then further tenfold dilutions were made up to -9 in
a reduced diluent (1/4 strength cysteinated Ringer diluent). 0.1 ml of each dilution
was spread on plates with different selective media to distinguish several bacterial
genera: total anaerobic counts (Wilkins-Chalgren agar), Bifidobacterium (Beerens'
medium), Clostridium spp. (TNS medium), and enterobacteria (McConkey agar). The tests
were duplicated for the first two media. Plates of the first three media were anaerobically
incubated for 5 to 7 d, and McConkey agar was aerobically incubated for 48 hours.
Colony counts were obtained and expressed as a log of the colony-forming units (CFU)
per gram of fresh faeces. Extemporarily, the fresh stool pH was measured by pH meter
(Bioblock, Illkirch, France).

Data analysis

Faecal bacteria concentrations were expressed as log colony forming unit (cfu)/g wet
weight. The results were expressed as means ± SEM for each period. Two-way ANOVA,
with time and treatment as factors, was used to compare bacterial concentrations,
pH, and faecal metabolites between the three periods. Following a significant F test
(P < 0.05), Newman-Keuls test was used to identify differences between individual
means. Symptoms experienced with scFOS were compared to those experienced with placebo
using Wilcoxon signed rank test.

Results

Bacterial counts and pH

Table 1 summarises bacterial counts and pH during basal, scFOS, and follow-up periods. Faecal
bifidobacteria counts were significantly increased during the scFOS period (9.17 ±
0.17 log cfu/g vs 8.52 ± 0.26 log cfu/g during the basal period; P < 0.05), and returned
to their baseline values during the follow-up period (8.37 ± 0.21 log cfu/g). Total
anaerobe counts did not change during scFOS period compared to the basal period, but
decreased in the follow-up period compared to the ingestion period (P < 0.05). Faecal
Clostridium counts were significantly increased during the follow-up period compared to the basal
and scFOS periods (P < 0.05). Faecal enterobacteria counts did not change during the
three periods. Faecal pH tended to decrease during scFOS and follow-up periods compared
to the basal period (P = 0.06).

Faecal neutral sterols and bile acids

Faecal cholesterol concentration increased during the scFOS period (8.18 ± 2.37 mg/g
dry matter vs 2.81 ± 0.94 mg/g dry matter during the basal period; P < 0.05)), and
returned to the baseline value during the follow-up period (2.87 ± 0.44 mg/g dry matter
(figure 1). However, no statistical differences were reported for coprostanol, cholestanol,
and ketones for the three periods (Table 2). Total neutral sterol concentrations and outputs did not change, but tended to increase
(p = 0.08) during the scFOS period.

Total bile acid concentrations and outputs were similar in the three periods. Concentrations
of secondary (lithocholic and deoxycholic acids) and primary bile acids (cholic and
chenodeoxycholic acids) did not change for the three periods (Table 2).

Stool weight and oro-fecal transit time

Stool wet weight, dry matter, and faecal water did not change throughout the study.
Oro-fecal transit time was not significantly modified by scFOS ingestion compared
to the basal and follow-up periods (Table 3).

Digestive tolerance

During scFOS ingestion, excessive flatus and bloating were significantly more frequent
when compared to the basal period (P < 0.05), but symptom intensity was very mild
(Table 4). Borborygmi and abdominal pain were not significantly different in all periods.

Discussion

The present experiment showed that four-week scFOS ingestion, with a dose of 8 g/d,
is well tolerated and leads to significant increase in faecal bifidobacteria and cholesterol
excretion in healthy elderly. The sc-FOS bifidogenic effect has been extensively demonstrated
in adults [6,26-28], but rarely in elderly [19,21,22]. Among the very few available studies about functional foods in elderly, one recent
double-blind trial testing a symbiotic (B. lactis BL-01, B. bifidum BB-02 and an inulin-based
prebiotic) also found promising results [29]. Significant increase in total bifidobacteria counts was indeed observed in the symbiotic
group compared with the placebo group.

We also found increasing Clostridium spp. after sc-FOS ingestion discontinuation. Clostridium spp. is a major component of normal anaerobic microflora and it can not be considered
as a deleterious or beneficial genus. Since some toxinogenic subspecies of Clostridium difficile are related to an increased risk of pseudo membranous colitis and/or infection in
older people, it would have been interesting to measure the sc-FOS effects on these
subspecies. However, we did not perform those analyses, for they were out of our study
scope. Further studies may investigate this point, using adequate measurement methods
for species concentrations and toxinogenic properties (cellular cultures, biomolecular
analysis). Culture-based enumeration of microbiota does not usually allow for bacterial
species measurement, but mainly bacterial genus.

In our study, several parameters were assessed with the objective to better understand
scFOS physiological effects in healthy elderly, such as transit time, stool characteristics,
and colonic environment. We did not find scFOS ingestion changed faecal weight and
oro-fecal transit time in elderly. Gibson et al. have shown that prebiotics can increase stool output: they studied 8 volunteers
under controlled diet, and showed that with 15 g/d fructo-oligosaccharides, stool
output significantly increased from 136 to 154 g/d [27]. Other two human studies did not demonstrate increasing stool output [2,30]. but the diet was not controlled in none of these studies, which may have hidden
any slight effect. In the study of Alles et al., 12 healthy subjects were given 4.8–19.2 g/d oligomate (52% galacto-oligosaccharides),
which did not result in any change in bowel habit. However, the subjects started with
unusually high faecal weights under controlled diet, 272 ± 26 g/d On the other hand,
studies using probiotics demonstrated bifidobacteria could reduce human colonic transit
time, but not all bifidobacteria strains have the same effects [31]. This specific strain-dependent effect could explain the reason why our prebiotic,
which stimulates global endogenous bifidobacteria, had no effect.

In our study, the microbial transformation of cholesterol into coprostanol was not
influenced by scFOS ingestion. Another study observed that sterol and fatty acid biohydrogenation
by intestinal microflora is altered by oligosaccharide fermentation [32]. Coprostanol production results from intestinal anaerobic bacteria action [33]. Concerning bile acid metabolism, no differences were observed during the three periods.
Furthermore, the use of poorly digestible carbohydrates in rats, hamsters and pigs
demonstrated that prevention of microbial conversion of bile acids depended on the
carbohydrate dose in the diet [23,34] This suggests that the low carbohydrate dose, 8 g/d scFOS, used in this experiment
is unable to modify microbial conversion of bile acids.

Endogenous or exogenous bile acids, as well as dietary cholesterol are carcinogenic
factors involved in colon cancer in laboratory animals [35,36] Various epidemiological studies suggest those steroids could also be involved in
colon cancer in men [12,37]. According to these studies, low scFOS dose ingestion by humans, which prevented
microbial conversion of cholesterol into cytotoxic molecule, (coprostanol, potentially
carcinogenetic), could be interesting for humans. In our study, the intake of 8 g/d
scFOS led to increasing faecal cholesterol. The mechanism of such increase could be
related to decreasing cholesterol bacterial transformation, although we failed to
find any significant sc-FOS effect on cholesterol bacterial metabolism. Moreover,
the low scFOS dose used in our study was also probably not sufficient to significantly
reduce microbial conversion of bile acids. However, in our previous study evaluating
a higher scFOS dose (12.5 g/d), we also failed to show any significant effect in bile
acids and neutral sterol [28]. These negative results could be explained by a questionable capacity of various
bifidobacteria to take up cholesterol into their cellular membrane [38].

Conclusion

Overall, we showed that 8 g/d scFOS ingested are well tolerated and led to significant
increase in faecal bifidobacteria in healthy elderly. Under our experimental conditions,
i.e. 8 g/d for 12 days, we failed to show any sc-FOS effects on OFTT, which is commonly
increased in elderly living in industrialised countries. We found significant change
in cholesterol metabolism, which could potentially exert protective action against
colon cancer; however, this finding warrants further studies.